CN105177164A - Molecular marker for early screening cervical cancer and detecting primers - Google Patents
Molecular marker for early screening cervical cancer and detecting primers Download PDFInfo
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- 238000012216 screening Methods 0.000 title claims abstract description 34
- 206010008342 Cervix carcinoma Diseases 0.000 title claims abstract description 25
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Abstract
本发明公开了一种用于宫颈癌早期筛查的分子标记和检测引物，所述的分子标记为分子标记JAM3-M4，其核苷酸序列如:SEQ？ID？No.1，扩增在序列的引物序列如下示：JAM3-M4的正向引物序列如SEQ？ID？No.6所示，反向引物序列如SEQ？ID？No.7所示。本发明首次发现JAM3基因的M4位点的甲基化程度与宫颈病变有关，可作为宫颈癌早期筛查的分子标记。本发明的用于检宫颈癌早期筛查的分子标记的引物及试剂盒。
The invention discloses a molecular marker and detection primers for early screening of cervical cancer. The molecular marker is molecular marker JAM3-M4, and its nucleotide sequence is as follows: SEQ? ID? No.1, the primer sequence for amplification in the sequence is as follows: the forward primer sequence of JAM3-M4 such as SEQ? ID? As shown in No.6, the reverse primer sequence is as SEQ? ID? Shown in No.7. The present invention first discovers that the degree of methylation of the M4 site of the JAM3 gene is related to cervical lesions, and can be used as a molecular marker for early screening of cervical cancer. The primer and the kit for molecular markers used for early screening of cervical cancer of the present invention.
Description
Technical field
The present invention relates to a kind of molecule marker for cervical cancer early screening and detect primer, belonging to molecular biology and medical field.
Background technology
Cervical cancer is one of common gynecologic malignant tumor, and the annual new cases in the whole world are about 528,000 example, and death about has 266, and 000 example, wherein about 85% occurs in developing country.The sickness rate of China remains high, and be 7.5/100,000, mortality ratio is 3.4/100,000.Lack the backward areas of reasonable examination at some, sickness rate even up to 81/100,000.According to a preliminary estimate, to the year two thousand thirty, the patient dying from cervical cancer every year about has 474, and 000 example, wherein may betide more than 95% and fall behind and developing country.Therefore, find the molecule marker of cervical cancer early screening, examination goes out precancerous lesions of uterine cervix and tumour patient, and enforcement is effectively early examined, early tool is significant.
At present, the cervical carcinoma screening strategy that what China adopted is is main contents with cervical liquid-based cells, local adopts HPV-DNA+ cytology United screening simultaneously.The national examination system of China's not yet Erecting and improving, and due to unbalancedness influence factors such as () regional economy, medical resource, public cognitives of examination, cause current examination shortage and examination excessively and deposit, treatment lack of standardization and over-treatment deposit.Cervical liquid-based cells existence 1, single application susceptibility not high (being about 50%-85%); 2, the specificity of ASCUS and LSIL is not high; 3, draw materials, film-making, diagosis level uneven, lack the defects such as normalized quality control.Therefore the inspection of single dependent cells easily causes false negative, and loss is high.HPV-DNA detects can significantly improve sensitivity, but often may detect transient HPV and infect, instead of has pathogenic high-risk HPV persistent infection.Therefore single dependence HPV examination, can cause excessively diagnosis and over-treatment.
DNA methylation is an important epigenetics mechanism.DNA methylation, namely non-methylated cytosine-phosphoric acid-guanine (Cytosine-phosphate-Guanosine, CpG) dinucleotides methylates, and is one of key mechanism of tumor suppressor gene inactivation.When tumor suppressor gene (TumorSuppressorGenes, TSG) is lost, suddenly change, the change such as to methylate causes normal function to be lost, the proliferation out of control formation malignant tumour of cell.Now clear and definite DNA methylation is one of three kinds of mechanism of TSG inactivation, and may be unique mechanism in some cases.Thus DNA methylation plays a significant role in cancer develops.As the earliest events that cancer occurs, DNA abnormal methylation detects can accomplish molecular diagnosis, for early diagnosis of cancer provides new approach before clinical manifestation or radiological evidence appear in patient.
Domestic and international investigator utilizes Relationship Between Methylation of Tumor Suppressor Gene to detect, in the research of cervical cancer early screening, achieve stem-winding progress, but same gene exists larger difference between difference research, analyzing its reason may be: 1, the specificity in gene methylation site; 2, the otherness of gene genetic background; 3, the heterogeneity of working method and experiment reagent.Therefore, this area is badly in need of finding the more effectively more stable biomarker being more easily applied to the specific early screening that methylates of clinical cervical cancer further.
Summary of the invention
For above-mentioned progress and prior art, present inventor is through retrieval and consult lot of documents and the data analysis to TheCancerGenomeAtlas (TCGA) platform, optimize multiple sites of several candidate gene, wherein multiple site for never reporting.By measuring, verify and analyzing, the M4 site of Late Cambrian JAM3 gene raises gradually with the methylated degree that increases the weight of of cervical lesions degree, can be used as the molecule marker of cervical cancer early screening, therefore, the invention provides a kind of molecule marker for cervical cancer early screening and detect primer.
To achieve these goals, the present invention adopts following technical scheme:
For a molecule marker for cervical cancer early screening, JAM3-M4, CADM1-M2, CADM1-M8, DAPK1-M2 or DAPK1-M3, nucleotide sequence is respectively:
JAM3-M4:CGCAGCCAGGGCTGGGACTCGGGCCTGGCTAGGGCGGGGGCCCTGGGACGCCCGGCAGTTGGACCGGGGCGGGGAGCTAATTTGGGATGGGGGGCCCGTCCCAGGCAGTGAGTCGGTCCCGGACAGCCGTCGGACCCC(SEQIDNO:1)，
CADM1-M2:GTCCTCCGCCACTTGTTGCTCCCGGGTCCTGCAGCTCTGGAGCTGCAAGGAGGGGCTTTGCAGGCTCAGAGCCCTGCTGCATCCCCTCCTGCATCGCCGCTCGCACCCCGCGGCCCC(SEQIDNO:2)，
CADM1-M8:GCCGCCGCACACTGGGATCCGCTCGGCAGCACTACACTCGCCATGTCGGGCACCTGCCTCAGACTGGCGGCGTTGGCTGCCTCCGGAGCCCGAGCGGACAGCTAATGAGA(SEQIDNO:3)，
DAPK1-M2:CGCTTGCAGGGTCCCCATTGGCCGCCTGCCGGCCGCCCTCCGCCCAAAAGGCGGCAAGGAGCCGAGAGGCTGCTTCGGAGTGTGAGGAGGACAGCCGGACCGAGCCAACGCCGGGGACTTTGTTCCCTCCGCGGAGGGGACTCGGCAACTCGCAGCGGCAG
(SEQIDNO:4)，
DAPK1-M3:GTCCCCATTGGCCGCCTGCCGGCCGCCCTCCGCCCAAAAGGCGGCAAGGAGCCGAGAGGCTGCTTCGGAGTGTGAGGAGGACAGCCGGACCGAGCCAACGCC(SEQIDNO:5)。
These sequence multidigits are in CpG island.The distribution of CpG dinucleotide in human genome very heterogeneity, and at some section genomic, CpG keeps or higher than normal probability, these sections are referred to as CpG island, the DNA fragmentation of 1 ~ 2kb in mammalian genes group, it is rich in non-methylated CpG amphiploid.CpG island is mainly positioned at promotor and the First Exon region of gene, about has the promotor of more than 60% gene to contain CpG island.GC content is greater than 50%, and length is more than 200bp.
CpG island is often positioned near transcription regulatory region, closely participates in the regulation and control of genetic expression and affects chromatinic structure.
Detect a primer for the above-mentioned molecule marker for cervical cancer early screening, described primer sequence shows respectively as follows:
The primer of JAM3-M4: MF:CGTAGTTAGGGTTGGGATTC (SEQIDNO:6)
MR:GAAATCCGACGACTATCCGA(SEQIDNO:7)，
The primer of CADM1-M2: MF:GTTTTTCGTTATTTGTTGTTTTC (SEQIDNO:8)
MR:GAAACCGCGAAATACGAACG(SEQIDNO:9)，
The primer of CADM1-M8: MF:GTCGTCGTATATTGGGATTC (SEQIDNO:10)
MR:TCTCATTAACTATCCGCTCG(SEQIDNO:11)，
The primer of DAPK1-M2: MF:CGTTTGTAGGGTTTTTATTGGTC (SEQIDNO:12)
MR:CTACCGCTACGAATTACCGA(SEQIDNO:13)，
The primer of DAPK1-M3: MF:GTTTTTATTGGTCGTTTGTC (SEQIDNO:14)
MR:GACGTTAACTCGATCCGACT(SEQIDNO:15)。
Above-mentioned primer is for the preparation of the application in the test kit of cervical cancer early screening or detecting sensor.
A kind of non-diagnostic object vitro detection sample JAM3-M4, the method for CADM1-M2, CADM1-M8, DAPK1-M2 or DAPK1-M3 methylation, step is as follows:
(1) extract the DNA of testing sample and carry out the modification that methylates;
(2) real-time quantitative methylation status of PTEN promoter amplification, β-actin:MF:TGGTGATGGAGGAGGTTTAGTAAGT, MR:AACCAATAAAACCTACTCCTCCCTTAA;
(3) formula is utilized: 2
[Ct (β-actin)-Ct (target)]the methylation of × 10,000 calculation sample gene.
Described PCR reaction conditions:
The methylation detecting said gene, except reaching the object of cervical cancer early screening, can also provide certain help for the biological function and meaning studying above gene.DNA methylation is the important mechanisms of tumour cancer suppressor gene inactivation, by after the methylation that detects said gene, can the expression of follow-up study corresponding gene, and the concrete mechanism of regulation and control biological behaviour.In addition, current Demethylation treatment, the activity recovering cancer suppressor gene also becomes the novel targets of clinical cancer therapy.
DNA methylation and methylation status of PTEN promoter (MethylationspecificPCR, MSP) principle: in normal people's genome, CpG site outside CpG island is normally methylated, and the CpG site in CpG island is in non-methylation state usually, this methylated form is with the stable heredity of cell fission.When tumour occurs, the non-methylation in the CpG site beyond cancer suppressor gene CpG island increases, and the CpG site in CpG island is hyper-methylation state, causes chromosome coiling degree to increase, Transcription inhibition, and genetic expression lacks.Methylation status of PTEN promoter PCR (MethylationspecificPCR, MSP) be study one of the most responsive experimental technique that methylates at present, methylating of minimum about 50pgDNA can be found, its ultimate principle is: single stranded DNA is after bisulphite modified, all unmethylated cytosine(Cyt) (cytosine, C) deamination changes uridylic (uracil into, U), and methylated cytosine(Cyt) remains unchanged in CpG site, therefore two are designed respectively to for methylating and the primer of non-methylated DNA fragments, can by (the Methylation that methylates by pcr amplification, M) with the non-(Unmethylation that methylates, U) DNA sequence dna makes a distinction.
Beneficial effect of the present invention:
Present inventor is through retrieval and consult lot of documents and the data analysis to TheCancerGenomeAtlas (TCGA) platform, optimizes multiple sites of several candidate gene, wherein has multiple site for never reporting.By measuring, verify and analyzing, the methylation in the M4 site of Late Cambrian JAM3 gene is relevant with cervical lesions, can be used as the molecule marker of cervical cancer early screening.
The present invention be used for cervical cancer early screening molecule marker primer and test kit, detection sensitivity, specificity, positive predictive value and negative predictive value, through the further checking of preliminary and a large amount of cervical exfoliated cell of clinical tissue sample, reach the requirement of clinical diagnosis.
JAM3-M4 is that ASCUS (being particularly less than the patient of 30 years old) and LSIL patient clinical usefulness are high for the patient of the shunting high-risk HPV positive and cytology screening results, also has certain prompting meaning in the CIN2 remaining dispute clinically in shunting.
The present invention be used for the molecule marker of cervical cancer early screening primer specificity strong, when adopting primer of the present invention to increase, amplification condition is easy to grope, and conveniently carries out specific amplification, is better than other primers.
Accompanying drawing explanation
Fig. 1 is 27 sites methylation differential expression level in cervical cancer tissues and normal control cervical tissue of methylation status of PTEN promoter (MSP) Preliminary detection 4 genes;
Fig. 2 is that-CADM1-M2, CADM1-M8, DAPK1-M2, DAPK1-M3 and JAM3-M4 diagnose and using value in examination in early days;
Fig. 3 is that JAM3-M4 is in the methylation level of uterine neck without knurl change-precancerous lesion-cancer patient cast-off cells;
Fig. 4 is the methylated clinical diagnosis usefulness of ROC tracing analysis JAM3-M4;
Fig. 5 is immunohistochemical staining and the Manganic pyrophosphate complex initiation of CIN2 case.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the invention will be further described.
1, the DNA extraction of cervical exfoliated cell:
Get the cervical exfoliated cell of patient, adopt QlAampDNAMiniKit (QiagenGmbH, Germany) test kit to extract, final gained DNA solution 80 μ L;
2, free serum DNA methylates modification:
Get the cast-off cells DNA that 1 μ g extracts, adopt QIAampEpiTectBis μ LfiteKits (QiagenGmbH, Germany) test kit to modify, final gained modifies rear DNA solution 30 μ L;
3, the optimization of real-time quantitative methylation status of PTEN promoter (QMSP) reaction system:
1). primer sequence in real time:
The primer of JAM3-M4: MF:CGTAGTTAGGGTTGGGATTC (SEQIDNO:6)
MR:GAAATCCGACGACTATCCGA(SEQIDNO:7)，
The primer of β-actin: MF:TGGTGATGGAGGAGGTTTAGTAAGT (SEQIDNO:16)
MR:AACCAATAAAACCTACTCCTCCCTTAA(SEQIDNO:17)。
2). the optimization of real-time PCR reactions system:
Real-time PCR reactions suit is from 1 × Power
greenPCRMasterMix (ABI, USA)
Real-time PCR reactions system:
Template: DNA after 1 μ L------modifies;
Primer: 1 μ L-------final concentration is 50nM;
MIX：10μL
ddH
2O：8μL
The detection sensitivity of detection method of the present invention, specificity, positive predictive value and negative predictive value and clinical samples are detected to checking and the assessment of application
1, the test kit used is:
(1) DNA extraction of cervical tissue and cast-off cells: QlAampDNAMiniKit (QiagenGmbH, Germany) test kit;
(2) DNA methylation is modified: QIAampEpiTectBis μ LfiteKitsQiagenGmbH, Germany) test kit;
(3) to methylate and non-methylate DNA standard substance: EpiTectPCRControlDNASet (QIAGEN, 59695);
(4) real-time PCR reactions suit: 1 × Power
greenPCRMasterMix (ABI, USA);
(5) PCR reaction suit: AmpliTaq
360MasterMix (ABI, USA);
2, PCR in real time condition:
Real-time PCR reactions condition:
3, Real time PCR results judges
(1) calculation formula of JAM3-M4 methylation is 2
[Ct (β-actin)-Ct (target)]× 10,000;
(2) the CT value of internal reference β-actin is greater than 32, and think that sample does not meet Quality Control, sample is defective;
(3) monitor the solubility curve of SYBRGreenPCR system, if solvent temperature deviation ± 2 DEG C of the solvent temperature of experiment sample and standard substance, then this sample is considered as invalid, need again detect.
One, 27 sites of methylation status of PTEN promoter (MSP) Preliminary detection 4 genes methylation differential expression level in cervical cancer tissues and normal control cervical tissue
Get the modification of carrying out methylating of 2 μ g clinical tissue sample DNAs and obtain reaction template; As seen from Figure 1, in representational cancer and normal cervical tissues, the visible obviously positive band of cancer, normally has no obvious band.Standard substance: M standard substance and U standard substance and unmodified template demonstrate the specificity of primer.Table 1 lists positive number of cases and the statistics P value in the most obvious 5 sites of difference based on the P value of two groups.Illustrate: through preliminary screening, have selected the research that 5 pairs of the most obvious MSP primers of difference carry out PCR in real time further.
Table 1
Primer sequence shows as follows:
The primer of JAM3-M4: MF:CGTAGTTAGGGTTGGGATTC (SEQIDNO:6)
MR:GAAATCCGACGACTATCCGA(SEQIDNO:7)，
The primer of JAM3-U4: UF:TGTAGTTAGGGTTGGGATTT (SEQIDNO:18) UR:CAAAATCCAACAACTATCCA (SEQIDNO:19),
The primer of CADM1-M2: MF:GTTTTTCGTTATTTGTTGTTTTC (SEQIDNO:8)
MR:GAAACCGCGAAATACGAACG(SEQIDNO:9)，
The primer of CADM1-U2: UF:GTTTTTTGTTATTTGTTGTTTTTG (SEQIDNO:20)
UR:AAAACCACAAAATACAAACA(SEQIDNO:21)，
The primer of CADM1-M8: MF:GTCGTCGTATATTGGGATTC (SEQIDNO:10)
MR:TCTCATTAACTATCCGCTCG(SEQIDNO:11)，
The primer of CADM1-U8: UF:GTTGTTGTATATTGGGATTT (SEQIDNO:22)
UR:TCTCATTAACTATCCACTCA(SEQIDNO:23)，
The primer of DAPK1-M2: MF:CGTTTGTAGGGTTTTTATTGGTC (SEQIDNO:12)
MR:CTACCGCTACGAATTACCGA(SEQIDNO:13)，
The primer of DAPK1-U2: UF:TGTTTGTAGGGTTTTTATTGGTTG (SEQIDNO:24)
UR:CCTACCACTACAAATTACCA(SEQIDNO:25)，
The primer of DAPK1-M3: MF:GTTTTTATTGGTCGTTTGTC (SEQIDNO:14)
MR:GACGTTAACTCGATCCGACT(SEQIDNO:15)，
The primer of DAPK1-U3: UF:TTTTATTGGTTGTTTGTTGG (SEQIDNO:26)
UR:CCCAACATTAACTCAATCCA(SEQIDNO:27)。
Two, at uterine neck without the checking in knurl change-precancerous lesion-cancer patient
1) 5 preliminary screening go out the checking-QMSP in site
Have collected the cast-off cells of uterine neck without knurl change-precancerous lesion-cancer patient in obstetrics and gynecology hospital outpatient service Vaginoscopy Room, Shandong, comprising chronic cervicitis 53 example, CIN159 example, CIN271 example, CIN363 example and SCC 20 example.Extract DNA from above-described sample after, the bisulphite modified template as QMSP is carried out to it.MSP has filtered out in cancer and healthy tissues the frequency difference degree that methylates maximum 5 sites--CADM1-M2, CADM1-M8, DAPK1-M2, DAPK1-M3 and JAM3-M4, verifies that it is diagnosed and the using value in examination in early days further by QMSP.Increase the weight of with lesion degree, the methylation of gene locus raises gradually, particularly evident with JAM3-M4, the obvious difference between diagnosis grouping, as shown in Figure 2.
2) Manganic pyrophosphate complex initiation checking JAM3-M4 is in the methylation level of uterine neck without knurl change-precancerous lesion-cancer patient cast-off cells
NILM (n=8) is selected at random from each grouping of sample of PCR in real time, CIN1 (n=10), CIN2QMSP feminine gender (QM-) (n=9), the CIN2QMSP positive (QM+) (n=8), CIN3 (n=10), cancer (n=9) sample carries out Manganic pyrophosphate complex initiation, further quantizes and verify (Fig. 3 A and B) the methylation level in JAM3-M4 site.In order to the interpretation of result with PCR in real time is before consistent, need the histodiagnosis of vaginoscope process to conform to clinical, the Manganic pyrophosphate complex initiation statistical analysis of CIN1-, CIN2, CIN3+ classification is as Fig. 3 C.
Three, the applied analysis of JAM3-M4 in clinical
1) JAM3-M4 is applied to separately clinical early screening
The method of inspection being widely used in early screening at present is clinically cytology and HPV, the power of test (susceptibility, specificity, positive predictive value and negative predictive value) of JAM3-M4 is compared with both, as table 2, prompting is compared with cytology with HPV, although susceptibility and negative predictive value have slight decline, but specificity and positive predictive value are significantly increased, show particularly evident when taking CIN3 as dividing value.
Table 2
2) application-United screening be combined with cytology and shunt examination
1. United screening namely in early days examination time, application cell and DNA methylation assay simultaneously, its power of test and as shown in table 3 with the contrast of at present conventional cytology and HPV United screening, although susceptibility and negative predictive value have slight decline, but specificity and positive predictive value are significantly increased, show particularly evident when taking CIN3 as dividing value.
Table 3
2. shunt examination namely in early days examination time, first application cell inspection, then to the patient of cytodiagnosis exception, carry out DNA methylation assay, it is shunted, determines whether carry out colposcopy and process, the shunting usefulness of JAM3-M4 and the contrast of shunting with conventional HPV as shown in table 4; In view of cytodiagnosis is that the specificity of HSIL is higher, having little significance of shunting, therefore JAM3-M4 to be shunted cytodiagnosis be that the patient of LSIL and ASCUS analyzes separately, as shown in table 5, for shunting cytodiagnosis be the effectiveness analysis of LSIL, specificity and positive predictive value significantly raise, and susceptibility and negative predictive value do not have impact; Be the effectiveness analysis of ASCUS for shunting cytodiagnosis, specificity and positive predictive value significantly improve, and susceptibility and negative predictive value have and to a certain degree decline.But the cytodiagnosis being less than to 30 years old (age bracket of HPV height infection rate) age is ASCUS patient, and 7 examples are the HPV positive, 5 examples are that CIN1-, JAM3-M4 are feminine gender, and prompting shunting meaning is larger.
Table 4
Table 5
3) application-United screening be combined with HPV and shunt examination
1. United screening namely in early days examination time, apply HPV and DNA methylation assay simultaneously, its power of test and as shown in table 6 with the contrast of at present conventional cytology and HPV United screening, although susceptibility has slight decline, but specificity, positive predictive value significantly improve, negative predictive value, in the slight rising of CIN3+/CIN1-and CIN3+/CIN2-diagnostics grouping, declines slightly at CIN2+/CN1-.
Table 6
2. shunt examination namely in early days examination time, first apply HPV check, then to the patient of the HPV positive, carry out DNA methylation assay, it is shunted, determines whether carry out colposcopy and process, the shunting usefulness of JAM3-M4 and the contrast of shunting with conventional cytology as shown in table 7; Specificity and positive predictive value significantly raise, and susceptibility slightly declines, and negative predictive value slightly rises.
Table 7
4) the methylated clinical diagnosis usefulness of ROC tracing analysis JAM3-M4
ROC curve is the conventional a kind of statistical method of Clinical Laboratory, can more different diagnostic test to disease identification ability.For different crowds---the case that the case that the case of Fig. 4 (A) all collections, the case of (B) cytodiagnosis exception, (C) cytodiagnosis are ASCUS, (D) cytodiagnosis are LSIL, the case of (E) HPV positive, by the methylated clinical diagnosis usefulness of ROC curve (area under curve the is measurement index) JAM3-4 that analyzed and researched; Area under curve prompting is all more meaningful to shunting clinically.
5) indicative significance of CIN2 is shunted
In view of above analysis, be that the clinical diagnosis usefulness of dividing value is all higher than CIN2+/CIN1-with CIN3.Assemble document analysis, CIN pathology can be divided into rf (CIN1 and CIN2) and transformant (CIN2 and CIN3) pathology.The CIN2 of rf and transformant cannot be distinguished by morphological observation, the guide of 2014 editions WHO proposes the dyeing carrying out immunohistochemical methods P16 with Histological section, according to positive feminine gender, CIN is split into HSIL and LSIL, immunohistochemical staining and Manganic pyrophosphate complex initiation such as Fig. 5 of representational CIN2 case show.In all CIN2 cases, the positive rate of P16 is 69.78%, and the rate that conforms to that the positive rate of JAM3-M4 is both 48.84%. is 60.47% (p=0.119), although statistical significance is not remarkable, or existence necessarily shunts indicative significance.
Claims (5)
1. for a molecule marker for cervical cancer early screening, it is characterized in that: described molecule marker is JAM3-M4, its nucleotide sequence as: shown in SEQIDNo.1.
2. test right requires a primer for the molecule marker for cervical cancer early screening described in 1, and described primer sequence is as follows: the forward primer sequence of JAM3-M4 is as shown in SEQIDNo.6, and reverse primer sequences is as shown in SEQIDNo.7.
3. primer according to claim 2 is for the preparation of the application in the test kit of cervical cancer early screening or detecting sensor.
4. a method for non-diagnostic object vitro detection sample JAM3-M4 methylation, is characterized in that: step is as follows:
(1) extract the DNA of testing sample and carry out the modification that methylates;
(2) real-time quantitative methylation status of PTEN promoter amplification, the forward primer sequence of β-actin is as shown in SEQIDNo.16, and reverse primer sequences is as shown in SEQIDNo.17;
(3) formula is utilized: 2
[Ct (β-actin)-Ct (target)]the methylation of × 10,000 calculation sample gene.
5. method according to claim 4, is characterized in that: described PCR reaction conditions:
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111793690A (en) * | 2020-07-23 | 2020-10-20 | 深圳市新合生物医疗科技有限公司 | DNA methylation qPCR kit for cervical cancer detection and use method thereof |
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| CN114507730B (en) * | 2020-11-16 | 2023-01-20 | 武汉艾米森生命科技有限公司 | Application of reagent for detecting gene methylation in cervical cancer diagnosis and kit |
| CN114093500A (en) * | 2021-10-12 | 2022-02-25 | 湖南索莱智能科技有限公司 | Method for screening cervical cancer by fusing multiple detection results |
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